1. Field of the Invention
The present invention relates generally to the fields of molecular virology and vaccinology. More specifically, the present invention relates to the attenuation of negative stranded RNA viruses by rearrangement of their genes and uses thereof.
2. Description of the Related Art
The order Mononegavirales is composed of four families, the Rhabdoviridae, the Paramyxoviridae, the Filoviridae and the Bornaviridae. The viruses in these families contain a single strand of non-segmented negative-sense RNA and are responsible for a wide range of significant diseases in fish, plants, and animals (Wagner, 1996). The expression of the genes encoded by these viruses is controlled at the level of transcription by the order of the genes on the genome relative to the single 3xe2x80x2 promoter. Gene order throughout the Mononegavirales is highly conserved: genes encoding products required in stoichiometric amounts for replication are always at or near the 3xe2x80x2 end of the genome while those whose products are needed in catalytic amounts are more promoter distal (Pringle and Easton, 1997).
Vesicular stomatitis virus (VSV) is the prototypic virus of the Rhabdoviridae. Its 11 kilobase genome has 5 genes which encode the 5 structural proteins of the virus; the nucleocapsid protein, N, which is required in stoichiometric amounts for encapsidation of the replicated RNA; the phosphoprotein, P, which is a cofactor of the RNA-dependent RNA polymerase, L; the matrix protein, M; and the attachment glycoprotein, G. The order of genes in the genome is 3xe2x80x2-N-P-M-G-L-5xe2x80x2 and previous studies have shown that expression is obligatorily sequential from a single 3xe2x80x2 promoter (Ball and White, 1976). Due to attenuation at each gene junction the 3xe2x80x2-most genes are transcribed more abundantly than those that are more promoter distal (Iverson and Rose, 1981).
In nature, VSV infects a wide range of animals of which horses, cattle, and domestic swine are the most economically important. Infection results in the appearance of lesions around the mouth, hooves, and udder teats and while seldom fatal it leads to a loss in meat and milk production along with the expense of quarantine and vaccination. There are two main VSV serotypes, Indiana (Ind) and New Jersey (NJ) and while these viruses are endemic in Central and South American countries, outbreaks do occur within the United States. A recent outbreak in the U.S. occurred in 1997 in horses, and was of the Ind serotype while previous cases identified in 1995 and 1982-1983 were of the NJ serotype. The ease with which these viruses are transmitted, and the similarity of their symptoms to those caused by foot-and-mouth disease virus in cattle and domestic swine, makes VSV a pathogen of concern to the agriculture industry.
Live attenuated viruses capable of replicating to generate protective humoral as well as cell mediated immune responses without producing disease manifestations have proven effective vaccines against viruses such as smallpox, yellow fever and poliomyelitis. The strategy for attenuation, however, has been empirical in most cases and not reproducible for general use. An additional consideration in the case of RNA viruses is that the high error rate of RNA dependent RNA polymerases, their lack of proof reading and the quasi-species nature of RNA virus populations (Domingo et al, 1996), make the use of live attenuated viruses for this large group of medically significant pathogens problematic. This is especially true if the vaccine virus is based on a limited number of single base changes as reversion to virulence is a potential problem. For example, only a few back mutations can restore virulence to the Sabin poliovirus type 3 vaccine strain (Wimmer et al., 1993).
The non-segmented negative strand RNA viruses of the family Mononegavirales possess an elegantly simple means of controlling the expression of their genes. The linear, single-stranded RNA genomes of this family encode five to ten genes, the order of which is highly conserved among all members. The prototype virus of this family is the Rhabdovirus, vesicular stomatitis virus (VSV). Transcription of the viral genome is carried out by the virus-encoded RNA dependent RNA polymerase. There is a single entry site on the linear genome for the RNA polymerase, yet the mRNAs of the virus are not produced in equimolar amounts.
Available evidence indicates that the linear order of the genes on the genome controls the levels of expression of individual genes. Transcription initiates at the single polymerase entry site at the 3xe2x80x2 terminus of the genome and is obligatorily processive (Ball and White, 1976). The level of expression of the individual genes a s monocistronic mRNAs is controlled by the dissociation, approximately 30% of the time, of the polymerase at each intergenic junction, as it traverses the genome in the 3xe2x80x2 to 5xe2x80x2 direction (Iverson and Rose, 1981). This mechanism of transcription results in sequentially decreasing amounts of the transcripts of each gene as a function of the distance of the gene from the 3xe2x80x2 terminus of the genome. Correspondingly, gene products needed in stoichiometric amounts to support replication, such as the nucleocapsid (N) protein, are encoded at or near the 3xe2x80x2 terminus in all cases and expressed in the highest molar amounts (Villarreal et al., 1976, Ball and White, 1976). Gene products needed in enzymatic amounts, such as the RNA polymerase are encoded most distal from the 3xe2x80x2 end. In all of the Mononegavirales, the polymerase gene is the 5xe2x80x2-most gene, and it is expressed in the lowest amount. Precise molar ratios of the proteins are required for optimal replication. For successful replication, proteins must be expressed in molar ratios that approximate those expressed normally from the genome (Pattnaik and Wertz, 1990).
Viruses of the family Mononegavirales do not undergo homologous genetic recombination (Pringle, 1987). Thus, other than defective interfering particles, which lack portions of the genome, variants of these viruses having the entire complement of genes in a rearranged format have not been observed in nature.
The prior art is deficient in the lack of effective means of increasing expression of a promoter distal gene in a virus of the order Mononegavirales and uses of such viruses. The present invention fulfills this long-standing need and desire in the art.
The non-segmented negative-strand RNA viruses (order Mononegavirales) comprise several important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site on the viral genome are transcribed at higher levels than those that occupy more distal positions. An infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) was manipulated to rearrange the order of four of the five viral genes, while leaving all other aspects of the viral nucleotide sequence unaltered. In one set of cDNA clones, the middle three genes (which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G) were rearranged into all six possible orders. In another set, the gene for the nucleocapsid protein N was moved away from its wild-type promoter-proximal position and placed second, third or fourth. In a final rearrangement, the G protein gene, which encodes the major surface antigen and the target for neutralizing antibodies, was put next to the promoter, in the position for maximum expression. Infectious viruses were recovered from each of these rearranged cDNAs and examined for their levels of gene expression and growth potential in cell culture, and their immunogenicity and virulence in mice. Rearrangement changed the expression levels of the encoded proteins and attenuated the viruses to different extents both in cultured cells and in mice. Increasing the expression of the G protein enhanced and accelerated the immune response in inoculated mice. Since the Mononegavirales do not undergo homologous recombination, gene rearrangement should be irreversible and thus provides a rational method for developing securely attenuated live vaccines against this type of virus.
In one embodiment of the present invention, there is provided a method of increasing expression of a promoter distal gene in a virus of the order Mononegavirales, comprising the step of rearranging gene order of the virus by moving the promoter distal gene toward a wild-type 3xe2x80x2 promoter proximal position site.
In another embodiment of the present invention, there is provided a recombinant virus of the order Mononegavirales having a rearranged genome, wherein the genome is rearranged by moving a promoter distal gene of the virus toward a wild type 3xe2x80x2 promoter proximal position site. Such recombinant virus can be used for accelerating and enhancing a protective immune response.
In still another embodiment of the present invention, there is provided a method of attenuating a virus of the order Mononegavirales by rearranging gene order of the virus by moving a gene away from its wild type position, or by rearranging gene order of the virus by moving an essential limiting factor gene away from its wild type 3xe2x80x2 promoter proximal position site.
In yet another embodiment of the present invention, there is provided a method of constructing an attenuated virus useful for a vaccine, comprising the steps of: rearranging the gene order of the virus by moving a gene away from its wild-type 3xe2x80x2 promoter proximal position site, wherein the gene is an essential limiting factor for genome replication; and placing a gene coding for an immune response inducing antigen in the position closest to the 3xe2x80x2 end of the gene order of the virus.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.